Uncontrolled tissue destruction by protease release plays a role in some pathological conditions, and it is hypothesized that an imbalance in the protease antiprotease system is an important contributing factor. Elastase is a protease that is capable of hydrolyzing the connective tissue component elastin. Elastase acts on a protein's nonterminal bonds adjacent to an aliphatic amino acid. Elastase is one of the matrix degrading enzymes found in plasma and is closely associated with elastin tissue destruction found in conditions such as abdominal aortic aneurysm (AAA) (Cohn, J. R., et al. Surg. Gynecol. Obstet. 1987; 164:355-358); emphysema (Snider, G. L., et al. Am. J. Resp. Crit. Case. Med. 1994; 150:5131-5137); pancreatitis (Dominguez-Muneoz, J. E., et al. Dig. Dis. Sci. 1993; 38:507-513; Stein, J., et al. Clin. Chem. 1996; 42:222-226); and inflammatory conditions (Andus, T., et al. Dig. Dis. Sci. 1993; 38:1638-1644), such as arthritis.
Since abdominal aortic aneurysms often go unnoticed until they become large enough to be felt as a mass or to cause symptoms, a method for the early diagnosis of an abdominal aortic aneurism is needed, and would be extremely useful for early treatment of aneurysms.
In order to understand the role of elastase in these conditions one must be able to determine elastase activity. There are a variety of ways to quantitatively measure proteases, such as elastase, such as the use of radiolabeled elastin (Bieger, W., and Sheele, G. (1980) Anal. Biochem. 104, 239-246), second enzyme-coupled elastin (Saunders, G.C., Svitra, Z., and Martinez, A. (1982) Anal. Biochem. 126, 122-130), and synthetic substrates (Gertler, A., and Hofmann, T. (1970) Can. J. Biochem. 48, 384-386; Bieth, J., Spiers, B., and Wermuth, C. G. (1974) Biochem. Med. 11, 350-357; Zhou, D., Niewiarowski, S., and Stewart, G. J. (1995) Anal. Biochem. 224, 436-437), radioimmunoassay (Murata, A., Ogawa, M., Fugimoto, K-I, Kitahara, T., Matsuda, Y., and Kosaki, G. (1983) Enzyme 30, 29-37) , zymography ( Brophy, C. M., Sumpio, B., Reilly, J. M., and Tilson, D. M. (1991) Surg. Res. Commun. 10, 315-321), and ELISA (Dreher, M., Gunzer, R., Helger, R., and Lang, H. (1989) Prog. Clin. Biol. Res. 308, 707-710). However, these techniques have limitations, such as low sensitivity, they are time-consuming and labor-intensive, and they are unable to distinguish between elastase and zymogen (inactive enzyme) There is therefore an outstanding need for a rapid and sensitive method of detecting and measuring elastase in a sample for use in the understanding of the role of elastase in connective tissue disorders and for diagnosing connective tissue disorders.
Habeeb, A. F. S. A., Anal.Biochem. 14:328-336 (1966 describes a method of detecting the presence of free amino groups in proteins using 2,4, 6-trinitrobenzenesulfonic acid. Hatakeyama, et al. Anal. Biochem. 204:181-184 (1992) describe an assay for detecting protease using succinylcasein as a substrate. Bubnis and Ofner, Anal. Biochem. 207:129-133 (1992) describe the detection of .epsilon.-amino groups in proteinaceous material using trinitrobenzenesulfonic acid. These references do not, however, address the need for a method of determining specific inhibitors of elastase.
The role of elastase in connective tissue disorders may be better understood through the identification of inhibitors of elastase, since the loss of inhibition of elastase is an important contributing factor for the development of connective tissue disorders. Elastase may be inactivated by naturally occurring inhibitors that block the active site of the enzyme by binding tightly to it. These naturally occurring elastase inhibitors are necessary to prevent elastase from unnecessarily degrading proteins which can lead to connective tissue destruction. Once inhibitors of elastase are identified, they may be used in the design of drugs for treating these connective tissue disorders. Thus, an outstanding need remains for a method of detecting inhibitors of proteases, such as elastase, for the development of inhibitors for diagnosing and treating connective tissue disorders.